Proteolically resistant cyclotides with angiotensin 1-7 like activity

ABSTRACT

Described herein is a novel cyclotide able to activate the unique receptor of angiotensin-(1-7) (AT1-7), the MAS1 receptor. This cyclotide may be used in the treatment of cancer and myocardial infarction.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/290,329, filed Feb. 2, 2016, the entire content of which is hereby incorporated by reference into the present disclosure.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. R01-GM090323, R01-GM113363, and R01-GM085006 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD OF DISCLOSURE

This disclosure relates generally to the field of therapeutic cyclic peptides, specifically for use in cancer treatment.

BACKGROUND

Modulation of the renin-angiotensin system (RAS) by angiotensin-(1-7) (AT1-7) is recently emerged as an attractive and novel chemotherapeutic and chemopreventive treatment for lung cancer. AT1-7 is a component of the renin-angiotensin system (RAS) with vasodilator, antiproliferative and anti-angiogenic properties. Recent studies have shown that AT 1-7 is able to reduce serum-stimulated growth of human lung cancer cells both in vitro and in vivo through activation of the unique AT1-7 receptor, MAS1. In results with human lung adenocarcinoma xenografts, AT1-7 was able to inhibit tumor growth through reduction in cyclooxygenase-2 (COX-2) activity and production of pro-inflammatory prostaglandins. In contrast, AT 1-7 had no effect on cyclooxygenase 1 (COX-1) activity in the same xenograft tumor. All these suggest that selective activation of the MAS1 receptor may represent a novel treatment for lung cancer through reduction of COX-2 activity. Unfortunately, AT1-7 has a limited clinical potential due to its unfavorable pharmacokinetic profile.

SUMMARY

Cyclotides are small globular microproteins typically ranging from 28 to 37 amino acids with a unique head-to-tail cyclized backbone topology that is stabilized by three disulfide bonds. The number and positions of cysteine residues are conserved throughout the family, forming the cyclic cystine-knot (CCK) motif that acts as a highly stable and versatile framework on which hyper-variable loops are arranged. This cyclic cystine-knot (CCK) framework provides an extremely rigid molecular scaffold with exceptional to resistance to thermal, chemical and biological degradation. Cyclotides can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.

Naturally-occurring cyclotides have shown to possess various pharmacologically-relevant activities, and have been reported to be able to cross mammalian cell membranes to antagonize intracellular protein-protein interactions in animal models. The main features of cyclotides are a remarkable stability due to the CCK framework, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics making them ideal substrates for molecular grafting of biological peptide epitopes.

The peptide AT1-7 is a hormone that in general counteracts the angiotensin II through its own signaling pathway involving the MAS receptor. Studies in animal models show that AT1-7 has ample therapeutic potential in cardiovascular disease, and more recently in lung cancer chemotherapy and chemoprevention. Despite its potential therapeutic value, AT1-7 does not offer ideal prospects for clinical use due to its poor pharmacodynamics and pharmacokinetic properties, mostly due to its rapid degradation in plasma.

Disclosed herein for the first time is the design and synthesis engineered cyclotides with similar biological activity to that of angiotensin polypeptides, e.g., the peptide AT1-7. The engineered cyclotide is able to fold correctly and shows high resistance to degradation by human serum therefore providing a promising new peptide-based lead for the treatment of cancer and myocardial infarction.

In one aspect, described herein are the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin-(1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT 1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide based in the treatment of cancer and myocardial infarction.

Additional advantages and other features of the present disclosure will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from the practice of the disclosure. The advantages of the disclosure may be realized and obtained as particularly pointed out in the appended claims.

As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 shows design of grafted cyclotide MCo-AT1-7 to activate the receptor MAS1. The upper part of the panel shows the primary and tertiary structures of the cyclotide MCoTI-I (structure is based on a homology model using the solution structure of MCoTI-II as template (PDB: 1IB9)), and the primary structure of peptide angiotensin 1-7 (AT1-7). The cyclized polypeptide is stabilized by three-disulfide bonds between cysteines as shown as lines connecting residues 4 and 21, 11 and 23, and 17 and 29. The lower part of the figure shows the strategy used to graft an AT1-7-derived peptide onto the loop 6 of cyclotide MCoTI-I. The AT1-7-derived peptide was linked to the cyclotide backbone through the side-side chains of the N- and C-terminal residues forming two isopeptide bonds. The sequences corresponding to the AT1-7-derived peptides are shown linked at the carboxy terminal end to residue 1 of MCoTI-I and at the amino terminal end to residue 32. Residue X represents L-2,3-diaminopropionic acid.

FIGS. 2A-2B show chemical synthesis and characterization of cyclotide MCo-AT1-7. FIG. 2A shows analytical High Performance Liquid Chromatography (HPLC) traces of for the linear thioester precursor, GSH-induced cyclization/folding crude after 24 hours and purified cyclotide. An asterisk indicates the desired peptide. FIG. 2B shows an analytical HPLC trace and an electrospray mass spectrometry (ES-MS) characterization of pure MCo-AT1-7. The molecular weight is 4234.2+/−0.6 Da and the expected average molecular weight is 4234.9 Da.

FIG. 3 shows proton nuclear magnetic resonance (1H-NMR) characterization of folded cyclotide MCo-AT1-7. Chemical shifts differences of the backbone, Nα-H and Hα protons between the common sequence (residues 1 through 32) of MCoTI-I and MCo-AT1-7.

FIG. 4 shows stability of cyclotides MCo-AT1-7 and MCoTI-I, and peptide AT1-7 and reduced linear MCo-AT1-7 precursor to human serum at 37° C. Undigested peptides were quantified by HPLC-MS/MS.

FIG. 5 shows biological activity of cyclotide MCo-AT1-7. MAS1 stably transfected CHO cells were tested using different concentrations of cyclotide MCo-AT1-7 and peptide AT1-7 in the absence or presence of MAS1 antagonist A779. The amount of intracellular NO was measured by fluorescence as described in the experimental section. Cyclotide MCoTI-I was used as negative control. The average of standard deviation of three experiments is shown.

FIG. 6 shows analytical reverse-phase C18-HPLC trace and ES-MS (deconvoluted) of linear Salkylated MCo-AT1-7. HPLC analysis was performed using a linear gradient of 0-70% solvent B over 30 minutes.

FIG. 7 shows stability of cyclotides MCo-AT1-7 and MCoTI-I; and peptides AT1-7 and reduced linear MCo-AT1-7 precursor to human serum at 37° C. Undigested peptides were quantified by HPLC-MS/MS.

FIGS. 8A-8B show the MCo-AT1-7 fold is similar to that of MCoTI-I. FIG. 8A shows the amide protons from the 1H{1H}-NOESY spectrum of MCo-AT1-7 are well dispersed (from 6.5 ppm to 9.8 ppm) and exhibit a large number of cross-peaks, which is indicative of a well-structured protein. FIG. 8B shows overlay of the 1H{1H}-NOESY spectra of MCo-AT1-7 (black) and MCoTI-I (gray) shows that these two spectra are very similar; chemical shift differences of amide and/or alpha protons of the proteins are less than 0.2 ppm.

DETAILED DESCRIPTION

This disclosure references various publications, patents and published patent specifications by an identifying citation or an Arabic number. The full citations for the disclosures referenced by an Arabic number are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.

Before the compositions and methods are described, it is to be understood that the invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd) edition (Cold Spring Harbor Laboratory Press (2002)); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Harlow and Lane, eds. (1999) Using Antibodies, A Laboratory Manual; Animal Cell Culture (R. I. Freshney, ed. (1987)); Zigova, Sanberg and Sanchez-Ramos, eds. (2002) Neural Stem Cells.

Definitions

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 0.1 or 1 where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X+0.1 or 1” or “X−0.1 or 1,” where appropriate. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

The term “isolated” as used herein with respect to proteins, polypeptides, cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other proteins, polypeptides, cells, nucleic acids, such as DNA or RNA, respectively, that are present in the natural source of the macromolecule. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together. A recombinant polynucleotide is a polynucleotide created or replicated using techniques (chemical or using host cells) other than by a cell in its native environment.

A “subject,” “individual” or “patient” is used interchangeably herein and refers to a vertebrate, for example a primate, a mammal or preferably a human. Mammals include, but are not limited to equines, canines, bovines, ovines, murines, rats, simians, humans, farm animals, sport animals and pets.

“Cells,” “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

“Amplify” “amplifying” or “amplification” of a polynucleotide sequence includes methods such as traditional cloning methodologies, PCR, ligation amplification (or ligase chain reaction, LCR) or other amplification methods. These methods are known and practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al. (1990) Mol. Cell Biol. 10(11):5977-5982 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.

The term “genotype” refers to the specific allelic composition of an entire cell, a certain gene or a specific polynucleotide region of a genome, whereas the term “phenotype’ refers to the detectable outward manifestations of a specific genotype.

As used herein, the term “gene” or “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. A gene may also refer to a polymorphic or a mutant form or allele of a gene.

“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present invention.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, last accessed on May 21, 2008. Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.

In one aspect, the term “equivalent” as it refers to polypeptides, proteins, or polynucleotides refers to polypeptides, oligopeptides, proteins, or polynucleotides, respectively having a sequence having a certain degree of homology or identity with the reference sequence of the polypeptides, proteins, or polynucleotides (or complement thereof when referring to polynucleotides). A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. In one aspect, an equivalent has at least 70%, or at least 75% or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, sequence identity to the reference polynucleotide or polypeptide. The term “equivalent” may also refer to a cyclotide equivalent that comprises a polypeptide that maintains a cysteine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.

Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40° C. in about 10×SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50° C. in about 6×SSC, and a high stringency hybridization reaction is generally performed at about 60° C. in about 1×SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg²⁺ normally found in a cell.

As used herein, the term “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes of clarity, when referring herein to a nucleotide of a nucleic acid, which can be DNA or an RNA, the terms “adenosine”, “cytidine”, “guanosine”, and “thymidine” are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.

The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

The terms “polypeptide,” “oligopeptide,” “protein,” and “peptide” are used interchangeably and refer to a polymer of amino acids of any length, held together by amide bonds. Polypeptides can have any primary, secondary, tertiary, or quaternary structure and may perform any function, known or unknown. A polypeptide can comprise standard amino acids, modified amino acids, unnatural amino acids, enantiomers, and analogs thereof. If present, modifications to the amino acids can be imparted before or after assembly, synthesis, or translation of the polypeptide. A polypeptide can be further modified by conjugation with a labeling component.

As used herein, the term “carrier” encompasses any of the standard carriers, such as a phosphate buffered saline solution, buffers, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Sambrook and Russell (2001), supra. Those skilled in the art will know many other suitable carriers for binding polynucleotides, or will be able to ascertain the same by use of routine experimentation. In one aspect of the invention, the carrier is a buffered solution such as, but not limited to, a PCR buffer solution.

A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

“Gene delivery,” “gene transfer,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection, sometimes called transduction), transfection, transformation or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). Unless otherwise specified, the term transfected, transduced or transformed may be used interchangeably herein to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a cell. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

A cell that “stably expresses” an exogenous polypeptide is one that continues to express a polypeptide encoded by an exogenous gene introduced into the cell either after replication if the cell is dividing or for longer than a day, up to about a week, up to about two weeks, up to three weeks, up to four weeks, for several weeks, up to a month, up to two months, up to three months, for several months, up to a year or more.

The term “express” refers to the production of a gene product. When used in reference to a cancer cell or a tumor cell, “express” may also refer to an increased or abnormal level of production of a gene product by the cancer or tumor cell relative to normal cells.

As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

A “gene product” or alternatively a “gene expression product” refers to the RNA generated when a gene is transcribed or the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

“Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.

The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

As used herein, a “vector” is a vehicle for transferring genetic material into a cell. Examples of such include, but are not limited to plasmids and viral vectors. A viral vector is a virus that has been modified to transduct genetic material into a cell. A plasmid vector is made by splicing a DNA construct into a plasmid. As is apparent to those of skill in the art, the appropriate regulatory elements are included in the vectors to guide replication and/or expression of the genetic material in the selected host cell.

A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, “retroviral mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. A “lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral Vectors, New York: Spring-Verlag Berlin Heidelberg.

In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5′ and/or 3′ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., a cell surface marker found on stem cells.

A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.

“Plasmids” used in genetic engineering are called “plasmic vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.

“Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. A eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples include simian, bovine, ovine, porcine, murine, rats, canine, equine, feline, avian, reptilian and human.

“Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells' genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 μm in diameter and 10 μm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to prokaryotic Cyanobacteria, bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

The term “propagate” means to grow a cell or population of cells. The term “growing” also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.

The term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

A “probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels are described and exemplified herein.

A “primer” is a short polynucleotide, generally with a free 3′ —OH group that binds to a target or “template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a “pair of primers” or a “set of primers” consisting of an “upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are well known in the art, and taught, for example in MacPherson et al. (1991) PCR: A Practical Approach, IRL Press at Oxford University Press. All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as “replication.” A primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et al., supra. The primers may optionall contain detectable labels and are exemplified and described herein.

As used herein, the term “detectable label” intends a directly or indirectly detectable compound or composition (other than a naturally occurring polynucleotide in its natural environment) that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluoresecence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^(th) ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.

As used herein, the term “purification marker” refers to at least one marker useful for purification or identification. A non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

The phrase “solid support” refers to non-aqueous surfaces such as “culture plates” “gene chips” or “microarrays.” Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are attached and arrayed on a gene chip for determining the DNA sequence by the hybridization approach, such as that outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of this invention can be modified to probes, which in turn can be used for detection of a genetic sequence. Such techniques have been described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. A probe also can be attached or affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Pat. No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

Various “gene chips” or “microarrays” and similar technologies are known in the art. Examples of such include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); a low-density array with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput, automated mass spectrometry systems with liquid-phase expression technology (Gene Trace Systems, Inc.); a thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput microarry system that can dispense from 12 to 64 spots onto multiple glass slides (Intelligent Bio-Instruments); Molecular Biology Workstation and NanoChip (Nanogen, Inc.); a microfluidic glass chip (Orchid Biosciences, Inc.); BioChip Arrayer with four PiezoTip piezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet (Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome); ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); and GenoSensor (Vysis, Inc.) as identified and described in Heller (2002) Annu. Rev. Biomed. Eng. 4:129-153. Examples of “gene chips” or a “microarrays” are also described in U.S. Patent Publication Nos.: 2007/0111322; 2007/0099198; 2007/0084997; 2007/0059769 and 2007/0059765 and U.S. Pat. Nos. 7,138,506; 7,070,740 and 6,989,267.

In one aspect, “gene chips” or “microarrays” containing probes or primers homologous to a polynucleotide described herein are prepared. A suitable sample is obtained from the patient, extraction of genomic DNA, RNA, protein or any combination thereof is conducted and amplified if necessary. The sample is contacted to the gene chip or microarray panel under conditions suitable for hybridization of the gene(s) or gene product(s) of interest to the probe(s) or primer(s) contained on the gene chip or microarray. The probes or primers may be detectably labeled thereby identifying the sequence(s) of interest. Alternatively, a chemical or biological reaction may be used to identify the probes or primers which hybridized with the DNA or RNA of the gene(s) of interest. The genotypes or phenotype of the patient is then determined with the aid of the aforementioned apparatus and methods.

A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. As used herein, “cancer” may refer both to precancerous cells as well as cancerous cells of a tumor such as a solid tumor.

The term “lung cancer” refers to a type of cancer that develops in the lung. The peptides of the present disclosure are useful for treatment of both primary lung cancers (carcinomas) and cancers that metastasized to the lung. Lung cancers include, but are not limited to small-cell lung carcinoma and non-small-cell lung carcinoma. MAS1 mRNA and protein have been detected in the lung cancer cell lines SK-LU-1, SK-MES-1, and A549 lung cancer cell lines. See Gallagher et al., Inhibition of human lung cancer cell growth by angiotensin-(1-7), Carcinogenesis 2004, 25(11):20145-52.

The term “breast cancer” refers to a type of cancer that develops in the breast. The peptides of the present disclosure are useful for treatment of breast cancers of any stage, including those positive for estrogen receptor, progesterone receptor, and/or HER2, as well as breast cancers “triple negative” for such receptors.

MAS1 refers to a proto-oncogene, its mRNA expression product, and its protein expression product (Accession Number NP_002368.1). The MAS1 gene encodes a class I seven-transmembrane G-protein-coupled receptor. The encoded protein is a receptor for angiotensin-(1-7) and preferentially couples to the Gq protein, activating the phospholipase C signaling pathway. The encoded protein may play a role in multiple processes including hypotension, smooth muscle relaxation and cardioprotection by mediating the effects of angiotensin-(1-7). The published protein sequence comprises (SEQ ID NO: 1): MDGSNVTSFVVEEPTNISTGRNASVGNAHRQIPIVHWVIMSISPVGFVENGILLWFLCFR MRRNPFTVYITHLSIADISLLFCIFILSIDYALDYELSSGHYYTIVTLSVTFLFGYNTGLYLL TAISVERCLSVLYPIWYRCHRPKYQSALVCALLWALSCLVTTMEYVMCIDREEESHSRN DCRAVIIFIAILSFLVFTPLMLVSSTILVVKIRKNTWASHSSKLYIVIMVTIIIFLIFAMPMRL LYLLYYEYWSTFGNLHHISLLFSTINSSANPFIYFFVGSSKKKRFKESLKVVLTRAFKDEM QPRRQKDNCNTVTVETVV. MAS1 protein has been detected in many types of cancers, including breast cancer, carcinoid, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and urothelial cancer (see, e.g. the Human Protein Atlas described in Ponten et al., The Human Protein Atlas as a proteomic resource for biomarker discovery, Journal of Internal Medicine 2011, November; 270(5): 428-46; Gallagher et al., Inhibition of human lung cancer cell growth by angiotensin-(1-7), Carcinogenesis 2004, 25(11):20145-52; Luo et al., Expression of MAS1 in breast cancer, Cancer Science 2015, September; 106(9): 1240-48).

A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. Studies in animal models generally may be used for guidance regarding effective dosages for treatment of diseases. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Thus, where a compound is found to demonstrate in vitro activity, for example as noted in the Tables discussed below one can extrapolate to an effective dosage for administration in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

As used herein, “treating” or “treatment” of a disease in a patient refers to (1) preventing the symptoms or disease from occurring in an animal that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this invention, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.

“Suppressing” or “inhibiting” tumor growth indicates a growth state that is curtailed compared to growth without any therapy. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a ³H-thymidine incorporation assay, or counting tumor cells. “Suppressing” means any or all of the following states: slowing, delaying, and “suppressing” tumor growth indicates a growth state that is curtailed when stopping tumor growth, as well as tumor shrinkage.

A “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be “positive” or “negative”.

Cyclotides are small globular microproteins (ranging from 28 to 37 amino acids) with a unique head-to-tail cyclized backbone, which is stabilized by disulfide bonds forming a cystine-knot motif. This cyclic cystine-knot (CCK) framework provides a rigid molecular platform with exceptional stability towards physical, chemical and biological degradation. These micro-proteins can be considered natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization, but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot. Furthermore, naturally occurring cyclotides have shown to possess various pharmacologically relevant activities, and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes.

Descriptive Embodiments

This disclosure provides a cyclotide comprising, or consisting essentially of, or yet further consisting of, a cyclotide backbone and an peptide comprising an angiotensin polypeptide or an equivalent thereof. In one aspect, the angiotensin polypeptide comprises, or consists essentially of, or yet further consists of a polypeptide of the group of an angiotensin1-1-7 polypeptide, an angiotensin 1-4 polypeptide, or an angiotensin 1-5 polypeptide, or an equivalent of each thereof. Non-limiting examples of the angiotensin polypeptide is a polypeptide from the group of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 (XRVIE), wherein X is the amino acid L-2,3-diaminopropionic acid, SEQ ID NO: 14 (XRVYIHE), wherein X is the amino acid L-2,3-diaminopropionic acid, SEQ ID NO: 15, SEQ ID NO: 16, or an equivalent of each thereof. Non-limiting examples of the cyclotide backbone is a polypeptide from the group of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NOS: 21 to 300, or an equivalent of each thereof, wherein the equivalent comprises a polypeptide that maintains a cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.

In another aspect, the cyclotide comprises, or alternatively consists essentially of, or yet further consists of an angiotensin-(1-7) (AT1-7) (amino acid sequence DRVYIHP, SEQ ID NO: 11) peptide grafted to a cyclotide backbone. In a further aspect, provided herein is a cyclotide from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or an equivalent of each thereof, wherein the equivalent comprises a polypeptide that maintains a cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.

Cyclotides are small globular microproteins (ranging from 28 to 37 amino acids) with a unique head-to-tail cyclized backbone, which is stabilized by disulfide bonds forming a cystine-knot motif. This cyclic cystine-knot (CCK) framework provides a rigid molecular platform with exceptional stability towards physical, chemical and biological degradation. These micro-proteins can be considered natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization, but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot. Furthermore, naturally occurring cyclotides have shown to possess various pharmacologically relevant activities, and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes.

The construction of the cyclotide is known in the art and has been described previously (see WO 2011/005598, which is incorporated herein for all purposes). Synthesis of peptides useful in the methods and compositions of the disclosure are also described herein and known in the art.

The preparation of a cyclotide may also entail the generation of a linear peptide that contains the desired cyclotide in a linear form, flanked by two peptide fragments that have affinity to each other so as to be capable of bringing two ends of the linear cyclotide together, facilitating cyclization. Accordingly, the present disclosure provides a polypeptide precursor for generating a cyclotide.

In one embodiment, the angiotensin peptide is grafted into loop 6 of the cyclotide. The cyclotide comprises a molecular framework comprising a sequence of amino acids forming a cyclic backbone wherein the cyclic backbone comprises sufficient disulfide bonds to confer knotted topology on the molecular framework or part thereof. Examples of cyclic backbone polypeptides are now in the art and described herein.

Reference herein to a “cyclotide backbone” includes a molecule comprising a sequence of amino acid residues or analogues thereof without free amino and carboxy termini. The cyclic backbone of the disclosure comprises sufficient disulfide bonds, or chemical equivalents thereof, to confer a knotted topology on the three-dimensional structure of the cyclic backbone. The term “cyclotide” as used herein refers to a peptide comprising a cyclic cystine knot motif defined by a cyclic backbone, at least two but preferably at least three disulfide bonds and associated beta strands in a particular knotted topology. The knotted topology involves an embedded ring formed by at least two backbone disulfide bonds and their connecting backbone segments being threaded by a third disulfide bond. However, a disulfide bond may be replaced or substituted by a mimic of a disulfide bond such as 1,4-disubstituted 1,2,3-triazoles introduced through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or 1,5-disubstituted 1,2,3-triazoles introduced through a ruthenium(II)-catalyzed method (RuAAC), or another form of bonding such as an amide bond, thioethers, diselenide bond, triazoles, hydrocarbon-based bridges, ionic bonds, hydrogen bonds, or hydrophobic bonds. In some embodiments, a cyclotide backbone comprises between about 20 and about 100, between about 25 and about 50, between about 27 and about 42, between about 30 and about 40, between about 32 and about 38, or between about 28 and 37 amino acid residues.

In some embodiments, the cyclotide backbone is comprised of, or alternatively consists essentially of MCoTI-I. The sequence of MCoTI-I is described FIG. 1. In one aspect, MCoTI-I comprises the sequence GGVCPKILQRCRRDSDCPGACICRGNGYCGSGSD (SEQ ID NO: 3), or an equivalent thereof. In another aspect, the sequence comprises GGBCPKILQRCRRDSDCPGACICRGAGYCGSGSD (SEQ ID NO: 2), or an equivalent thereof. In some aspects, two residues are removed from the carboxy terminal end so that the sequence of MCoTI-I comprises or consists essentially of GGBCPKILQRCRRDSDCPGACICRGAGYCGSG (SEQ ID NO: 4) or GGVCPKILQRCRRDSDCPGACICRGNGYCGSG (SEQ ID NO: 5). In further aspect, residue B in the above sequences represents asparagine or aspartic acid.

Additional cyclotides useful in the peptides, methods, and compositions described herein are known in the art and non-limiting examples include, the cyclotides listed in Table 1 below. In some embodiments, the cyclotide backbone is derived from, comprises, or alternatively consists essentially of one or more of the sequences listed in Table 1 (SEQ ID NOS: 21-300). In some aspects, residue X in the amino acid sequences of Table 1 represents one or more unnatural amino acids.

TABLE 1  SEQ ID Parental Cyclotide NO: Cyclotide Backbone Sequence kalata_B1 21 GLPVCGETCVGGTCNTPGCTCSWPVCTRN cycloviolacin_O1 22 GIPCAESCVYIPCTVTALLGCSCSNRVCYN kalata_B2 23 GLPVCGETCFGGTCNTPGCSCTWPICTRD Palicourein 24 GDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN vhr1 25 GIPCAESCVWIPCTVTALLGCSCSNKVCYN tricyclon_A 26 GGTIFDCGESCFLGTCYTKGCSCGEWKLCYGTN circulin_A 27 GIPCGESCVWIPCISAALGCSCKNKVCYRN N-KB1-C 28 GLPVCGETCVGGTCNTPGCTCSWPVCTRN Ac-KB1-C 29 GLPVCGETCVGGTCNTPGCTCSWPVCTRN N-KB1-Am 30 GLPVCGETCVGGTCNTPGCTCSWPVCTRN Ac-KB1-Am 31 GLPVCGETCVGGTCNTPGCTCSWPVCTRN Ac-[desGly]-KB1-Am 32 LPVCGETCVGGTCNTPGCTCSWPVCTRN kalata_b1-1 33 TCVGGTCNTPGCTCSWPVCTRNLPVCG kalata_b1-2 34 GTCNTPGCTCSWPVCTRNGLPVCGETCVG kalata_b1-3 35 GCTCSWPVCTRNGLPVCGETCVGGTCN kalata_b1-4 36 CSWPVCTRNGLPVCGETCVGGTCNTPGC kalata_b1-5 37 VCTRNGLPVCGETCVGGTCNTPGCTCS kalata_b1-6a 38 VCGETCVGGTCNTPGCTCSWPVCT kalata_b1-6b 39 RNGLPVCGETCVGGTCNTPGCTCSWPVCT cycloviolacin_O2 40 GIPCGESCVWIPCISSAIGCSCKSKVCYRN des(24-28)kB1 41 VCGETCVGGTCNTPGCTCSWPVCT [Ala1,15]kB1 42 GLPVAGETCVGGTCNTPGATCSWPVCTRN kalata_B6 43 GLPTCGETCFGGTCNTPGCSCSSWPICTRN kalata_B3 44 GLPTCGETCFGGTCNTPGCTCDPWPICTRD kalata_B7 45 GLPVCGETCTLGTCYTQGCTCSWPICKRN cycloviolacin_O8 46 GTLPCGESCVWIPCISSVVGCSCKSKVCYKN cycloviolacin_O11 47 GTLPCGESCVWIPCISAVVGCSCKSKVCYKN kalata_B4 48 GLPVCGETCVGGTCNTPGCTCSWPVCTRD vodo_M 49 GAPICGESCFTGKCYTVQCSCSWPVCTRN cyclopsychotride_A 50 SIPCGESCVFIPCTVTALLGCSCKSKVCYKN cycloviolacin_H1 51 GIPCGESCVYIPCLTSAIGCSCKSKVCYRN cycloviolacin_O9 52 GIPCGESCVWIPCLTSAVGCSCKSKVCYRN vico_A 53 GSIPCAESCVYIPCFTGIAGCSCKNKVCYYN vitri_A 54 GIPCGESCVWIPCITSAIGCSCKSKVCYRN kalata_S 55 GLPVCGETCVGGTCNTPGCSCSWPVCTRN cycloviolacin_O12 56 GLPICGETCVGGTCNTPGCSCSWPVCTRN vodo_N 57 GLPVCGETCTLGKCYTAGCSCSWPVCYRN vico_B 58 GSIPCAESCVYIPCITGIAGCSCKNKVCYYN kalata_B1_IIa 59 GLPVCGETCVGGTCNTPGCTCSWPVCTRN Hypa_A 60 GIPCAESCVYIPCTITALLGCSCKNKVCYN circulin_B 61 GVIPCGESCVFIPCISTLLGCSCKNKVCYRN circulin_C 62 GIPCGESCVFIPCITSVAGCSCKSKVCYRN circulin_D 63 KIPCGESCVWIPCVTSIFNCKCENKVCYHD circulin_E 64 KIPCGESCVWIPCLTSVFNCKCENKVCYHD circulin_F 65 AIPCGESCVWIPCISAAIGCSCKNKVCYR cycloviolacin_O4 66 GIPCGESCVWIPCISSAIGCSCKNKVCYRN cycloviolacin_O3 67 GIPCGESCVWIPCLTSAIGCSCKSKVCYRN cycloviolacin_O5 68 GTPCGESCVWIPCISSAVGCSCKNKVCYKN cycloviolacin_O6 69 GTLPCGESCVWIPCISAAVGCSCKSKVCYKN cycloviolacin_O7 70 SIPCGESCVWIPCTITALAGCKCKSKVCYN cycloviolacin_O10 71 GIPCGESCVYIPCLTSAVGCSCKSKVCYRN kalata_B5 72 GTPCGESCVYIPCISGVIGCSCTDKVCYLN varv_peptide_B 73 GLPVCGETCFGGTCNTPGCSCDPWPMCSRN varv_peptide_C 74 GVPICGETCVGGTCNTPGCSCSWPVCTRN varv_peptide_D 75 GLPICGETCVGGSCNTPGCSCSWPVCTRN varv_peptide_F 76 GVPICGETCTLGTCYTAGCSCSWPVCTRN varv_peptide_G 77 GVPVCGETCFGGTCNTPGCSCDPWPVCSRN varv_peptide_H 78 GLPVCGETCFGGTCNTPGCSCETWPVCSRN cycloviolin_A 79 GVIPCGESCVFIPCISAAIGCSCKNKVCYRN cycloviolin_B 80 GTACGESCYVLPCFTVGCTCTSSQCFKN cycloviolin_C 81 GIPCGESCVFIPCLTTVAGCSCKNKVCYRN cycloviolin_D 82 GFPCGESCVFIPCISAAIGCSCKNKVCYRN violapeptide_1 83 GLPVCGETCVGGTCNTPGCSCSRPVCTXN vhl-1 84 SISCGESCAMISFCFTEVIGCSCKNKVCYLN Vontr_Protein 85 ALETQKPNHLEEALVAFAKKGNLGGLP hcf-1 86 GIPCGESCHYIPCVTSAIGCSCRNRSCMRN htf-1 87 GIPCGDSCHYIPCVTSTIGCSCTNGSCMRN Oantr_protein 88 GVKSSETTLMFLKEMQLKLP vhl-2 89 GLPVCGETCFTGTCYTNGCTCDPWPVCTRN cycloviolacin_H3 90 GLPVCGETCFGGTCNTPGCICDPWPVCTRN cycloviolacin_H2 91 SAIACGESCVYIPCFIPGCSCRNRVCYLN Hyfl_A 92 SISCGESCVYIPCTVTALVGCTCKDKVCYLN Hyfl_B 93 GSPIQCAETCFIGKCYTEELGCTCTAFLCMKN Hyfl_C 94 GSPRQCAETCFIGKCYTEELGCTCTAFLCMKN Hyfl_D 95 GSVPCGESCVYIPCFTGIAGCSCKSKVCYYN Hyfl_E 96 GEIPCGESCVYLPCFLPNCYCRNHVCYLN Hyfl_F 97 SISCGETCTTFNCWIPNCKCNHHDKVCYWN Hyfl_G_(partial) 98 CAETCVVLPCFIVPGCSCKSSVCYFN Hyfl_H_(partial) 99 CAETCIYIPCFTEAVGCKCKDKVCYKN Hyfl_I 100 GIPCGESCVFIPCISGVIGCSCKSKVCYRN Hyfl_J 101 GIACGESCAYFGCWIPGCSCRNKVCYFN Hyfl_K 102 GTPCGESCVYIPCFTAVVGCTCKDKVCYLN Hyfl_L 103 GTPCAESCVYLPCFTGVIGCTCKDKVCYLN Hyfl_M 104 GNIPCGESCIFFPCFNPGCSCKDNLCYYN Hyfl_N_(partial) 105 CGETCVILPCISAALGCSCKDTVCYKN Hyfl_O_(partial) 106 CGETCVIFPCISAAFGCSCKDTVCYKN Hyfl_P 107 GSVPCGESCVWIPCISGIAGCSCKNKVCYLN Hymo_A_(partial) 108 CGETCLFIPCIFSVVGCSCSSKVCYRN Hymo_B_(partial) 109 CGETCVTGTCYTPGCACDWPVCKRD Hyst_A_(partial) 110 CGETCIWGRCYSENIGCHCGFGICTLN Hyve_A_(partial) 111 CGETCLFIPCLTSVFGCSCKNRGCYKI Hyca_A_(partial) 112 CGETCVVDTRCYTKKCSCAWPVCMRN Hyde_A_(partial) 113 CVWIPCISAAIGCSCKSKVCYRN Hyen_A_(partial) 114 CGESCVYIPCTVTALLGCSCKDKVCYKN Hyen_B_(partial) 115 CGETCKVTKRCSGQGCSCLKGRSCYD Hyep_A_(partial) 116 CGETCVVLPCFIVPGCSCKSSVCYFN Hyep_B_(partial) 117 CGETCIYIPCFTEAVGCKCKDKVCYKN tricyclon_B 118 GGTIFDCGESCFLGTCYTKGCSCGEWKLCYGEN kalata_B8 119 GSVLNCGETCLLGTCYTTGCTCNKYRVCTKD cycloviolacin_H4 120 GIPCAESCVWIPCTVTALLGCSCSNNVCYN cycloviolacin_O13 121 GIPCGESCVWIPCISAAIGCSCKSKVCYRN violacin_A 122 SAISCGETCFKFKCYTPRCSCSYPVCK cycloviolacin_O14 123 GSIPACGESCFKGKCYTPGCSCSKYPLCAKN cycloviolacin_O15 124 GLVPCGETCFTGKCYTPGCSCSYPICKKN cycloviolacin_O16 125 GLPCGETCFTGKCYTPGCSCSYPICKKIN cycloviolacin_O17 126 GIPCGESCVWIPCISAAIGCSCKNKVCYRN cycloviolacin_O18 127 GIPCGESCVYIPCTVTALAGCKCKSKVCYN cycloviolacin_O19 128 GTLPCGESCVWIPCISSVVGCSCKSKVCYKD cycloviolacin_O20 129 GIPCGESCVWIPCLTSAIGCSCKSKVCYRD cycloviolacin_O21 130 GLPVCGETCVTGSCYTPGCTCSWPVCTRN cycloviolacin_O22 131 GLPICGETCVGGTCNTPGCTCSWPVCTRN cycloviolacin_O23 132 GLPTCGETCFGGTCNTPGCTCDSSWPICTHN cycloviolacin_O24 133 GLPTCGETCFGGTCNTPGCTCDPWPVCTHN cycloviolacin_O25 134 DIFCGETCAFIPCITHVPGTCSCKSKVCYFN [P20D, V21K]-kalata_B1 135 GLPVCGETCVGGTCNTPGCTCSWDKCTRN [W19K,_P20N,_V21K]- 136 GLPVCGETCVGGTCNTPGCTCSKNKCTRN kalata_B1 [Glu(Me)]cyO2 137 GIPCGXSCVWIPCISSAIGCSCKSKVCYRN [Lys(Ac)]2cyO2 138 GIPCGESCVWIPCISSAIGCSCXSXVCYRN [Arg(CHD)]cyO2 139 GIPCGESCVWIPCISSAIGCSCKSKVCYXN ([Lys(Ac)]2[Arg(CHD)]) 140 GIPCGESCVWIPCISSAIGCSCXSXVCYXN cyO2 kalata_B1_oia 141 GLPVCGETCVGGTCNTPGCTCSWPVCTRN kalata_B1_nfk 142 GLPVCGETCVGGTCNTPGCTCSWPVCTRN kalata_B2_nfk 143 GLPVCGETCFGGTCNTPGCSCTWPICTRD kalata_B2_kyn 144 GLPVCGETCFGGTCNTPGCSCTWPICTRD kalata_B9 145 GSVFNCGETCVLGTCYTPGCTCNTYRVCTKD kalata_B10 146 GLPTCGETCFGGTCNTPGCSCSSWPICTRD kalata_B10_oia 147 GLPTCGETCFGGTCNTPGCSCSSWPICTRD kalata_B11 148 GLPVCGETCFGGTCNTPGCSCTDPICTRD kalata_B12 149 GSLCGDTCFVLGCNDSSCSCNYPICVKD kalata_B13 150 GLPVCGETCFGGTCNTPGCACDPWPVCTRD kalata_B14 151 GLPVCGESCFGGTCNTPGCACDPWPVCTRD kalata_B15 152 GLPVCGESCFGGSCYTPGCSCTWPICTRD kalata_B16 153 GIPCAESCVYIPCTITALLGCKCQDKVCYD kalata_B17 154 GIPCAESCVYIPCTITALLGCKCKDQVCYN Amrad_5 155 CGETCVGGTCNTPGCTCSWPVCRRKRRR Amrad_9 156 CGETCRRKRRRCNTPGCTCSWPVCTRNGLPV Amrad_11 157 CGETCVGGTCNTRRKRRRGCTCSWPVCTRNGLPV Amrad_17 158 CGETCVGGTCNTPGCTCRRKRRRVCTRNGLPV Amrad_7 159 CGETCVGGTCNTPGCTCRRKRRRCTRNGLPV Amrad_8 160 CGETCVGGTCRRKRRRCTCSWPVCTRNGLPV kalata_B18 161 GVPCAESCVYIPCISTVLGCSCSNQVCYRN PS-1 162 GFIPCGETCIWDKTCHAAGCSCSVANICVRN CD-1 163 GADGFCGESCYVIPCISYLVGCSCDTIEKVCKRN cycloviolacin_Y1 164 GGTIFDCGETCFLGTCYTPGCSCGNYGFCYGTN cycloviolacin_Y2 165 GGTIFDCGESCFLGTCYTAGCSCGNWGLCYGTN cycloviolacin_Y3 166 GGTIFDCGETCFLGTCYTAGCSCGNWGLCYGTN cycloviolacin_Y4 167 GVPCGESCVFIPCITGVIGCSCSSNVCYLN cycloviolacin_Y5 168 GIPCAESCVWIPCTVTALVGCSCSDKVCYN vibi_A 169 GLPVCGETCFGGTCNTPGCSCSYPICTRN vibi_B 170 GLPVCGETCFGGTCNTPGCTCSYPICTRN vibi_C 171 GLPVCGETCAFGSCYTPGCSCSWPVCTRN vibi_D 172 GLPVCGETCFGGRCNTPGCTCSYPICTRN vibi_E 173 GIPCAESCVWIPCTVTALIGCGCSNKVCYN vibi_F 174 GTIPCGESCVFIPCLTSALGCSCKSKVCYKN vibi_G 175 GTFPCGESCVFIPCLTSAIGCSCKSKVCYKN vibi_H 176 GLLPCAESCVYIPCLTTVIGCSCKSKVCYKN vibi_I 177 GIPCGESCVWIPCLTSTVGCSCKSKVCYRN vibi_J 178 GTFPCGESCVWIPCISKVIGCACKSKVCYKN vibi_K 179 GIPCGESCVWIPCLTSAVGCPCKSKVCYRN Viba_2 180 GIPCGESCVYLPCFTAPLGCSCSSKVCYRN Viba_5 181 GIPCGESCVWIPCLTATIGCSCKSKVCYRN Viba_10 182 GIPCAESCVYLPCVTIVIGCSCKDKVCYN Viba_12 183 GIPCAESCVWIPCTVTALLGCSCKDKVCYN Viba_14 184 GRLCGERCVIERTRAWCRTVGCICSLHTLECVRN Viba_17 185 GLPVCGETCVGGTCNTPGCGCSWPVCTRN Viba_15 186 GLPVCGETCVGGTCNTPGCACSWPVCTRN mram_1 187 GSIPCGESCVYIPCISSLLGCSCKSKVCYKN mram_2 188 GIPCAESCVYIPCLTSAIGCSCKSKVCYRN mram_3 189 GIPCGESCVYLPCFTTIIGCKCQGKVCYH mram_4 190 GSIPCGESCVFIPCISSVVGCSCKNKVCYKN mram_5 191 GTIPCGESCVFIPCLTSAIGCSCKSKVCYKN mram_6 192 GSIPCGESCVYIPCISSLLGCSCESKVCYKN mram_7 193 GSIPCGESCVFIPCISSIVGCSCKSKVCYKN mram_8 194 GIPCGESCVFIPCLTSAIGCSCKSKVCYRN mram_9 195 GVPCGESCVWIPCLTSIVGCSCKNNVCTLN mram_10 196 GVIPCGESCVFIPCISSVLGCSCKNKVCYRN mram_11 197 GHPTCGETCLLGTCYTPGCTCKRPVCYKN mram_12 198 GSAILCGESCTLGECYTPGCTCSWPICTKN mram_13 199 GHPICGETCVGNKCYTPGCTCTWPVCYRN mram_14 200 GSIPCGEGCVFIPCISSIVGCSCKSKVCYKN Viba_1 201 GIPCGEGCVYLPCFTAPLGCSCSSKVCYRN Viba_3 202 GIPCGESCVWIPCLTAAIGCSCSSKVCYRN Viba_4 203 GVPCGESCVWIPCLTSAIGCSCKSSVCYRN Viba_6 204 GIPCGESCVLIPCISSVIGCSCKSKVCYRN Viba_7 205 GVIPCGESCVFIPCISSVIGCSCKSKVCYRN Viba_8 206 GAGCIETCYTFPCISEMINCSCKNSRCQKN Viba_9 207 GIPCGESCVWIPCISSAIGCSCKNKVCYRK Viba_11 208 GIPCGESCVWIPCISGAIGCSCKSKVCYRN Viba_13 209 TIPCAESCVWIPCTVTALLGCSCKDKVCYN Viba_16 210 GLPICGETCTLGTCYTVGCTCSWPICTRN [G1A]kalata_B1 211 ALPVCGETCVGGTCNTPGCTCSWPVCTRN [L2A]kalata_B1 212 GAPVCGETCVGGTCNTPGCTCSWPVCTRN [P3A]kalata_B1 213 GLAVCGETCVGGTCNTPGCTCSWPVCTRN [V4A]kalata_B1 214 GLPACGETCVGGTCNTPGCTCSWPVCTRN [G6A]kalata_B1 215 GLPVCAETCVGGTCNTPGCTCSWPVCTRN [E7A]kalata_B1 216 GLPVCGATCVGGTCNTPGCTCSWPVCTRN [T8A]kalata_B1 217 GLPVCGEACVGGTCNTPGCTCSWPVCTRN [V10A]kalata_B1 218 GLPVCGETCAGGTCNTPGCTCSWPVCTRN [G11A]kalata_B1 219 GLPVCGETCVAGTCNTPGCTCSWPVCTRN [G12A]kalata_B1 220 GLPVCGETCVGATCNTPGCTCSWPVCTRN [T13A]kalata_B1 221 GLPVCGETCVGGACNTPGCTCSWPVCTRN [N15A]kalata_B1 222 GLPVCGETCVGGTCATPGCTCSWPVCTRN [T16A]kalata_B1 223 GLPVCGETCVGGTCNAPGCTCSWPVCTRN [P17A]kalata_B1 224 GLPVCGETCVGGTCNTAGCTCSWPVCTRN [G18A]kalata_B1 225 GLPVCGETCVGGTCNTPACTCSWPVCTRN [T20A]kalata_B1 226 GLPVCGETCVGGTCNTPGCACSWPVCTRN [S22A]kalata_B1 227 GLPVCGETCVGGTCNTPGCTCAWPVCTRN [W23A]kalata_B1 228 GLPVCGETCVGGTCNTPGCTCSAPVCTRN [P24A]kalata_B1 229 GLPVCGETCVGGTCNTPGCTCSWAVCTRN [V25A]kalata_B1 230 GLPVCGETCVGGTCNTPGCTCSWPACTRN [T27A]kalata_B1 231 GLPVCGETCVGGTCNTPGCTCSWPVCARN [R28A]kalata_B1 232 GLPVCGETCVGGTCNTPGCTCSWPVCTAN [N29A]kalata_B1 233 GLPVCGETCVGGTCNTPGCTCSWPVCTRA Cter_A 234 GVIPCGESCVFIPCISTVIGCSCKNKVCYRN Cter_B 235 GVPCAESCVWIPCTVTALLGCSCKDKVCYLN hcf-1_variant 236 GIPCGESCHIPCVTSAIGCSCRNRSCMRN Vpl-1 237 GSQSCGESCVLIPCISGVIGCSCSSMICYFN Vpf-1 238 GIPCGESCVFIPCLTAAIGCSCRSKVCYRN cO31 239 GLPVCGETCVGGTCNTPGCSCSIPVCTRN cO28 240 GLPVCGETCVGGTCNTPGCSCSWPVCFRD cO32 241 GAPVCGETCFGGTCNTPGCTCDPWPVCTND cO33 242 GLPVCGETCVGGTCNTPYCTCSWPVCTRD cO34 243 GLPVCGETCVGGTCNTEYCTCSWPVCTRD cO35 244 GLPVCGETCVGGTCNTPYCFCSWPVCTRD cO29 245 GIPCGESCVWIPCISGAIGCSCKSKVCYKN cO30 246 GIPCGESCVWIPCISSAIGCSCKNKVCFKN cO26 247 GSIPACGESCFRGKCYTPGCSCSKYPLCAKD cO27 248 GSIPACGESCFKGWCYTPGCSCSKYPLCAKD Globa_F 249 GSFPCGESCVFIPCISAIAGCSCKNKVCYKN Globa_A 250 GIPCGESCVFIPCITAAIGCSCKTKVCYRN Globa_B 251 GVIPCGESCVFIPCISAVLGCSCKSKVCYRN Globa_D 252 GIPCGETCVFMPCISGPMGCSCKHMVCYRN Globa_G 253 GVIPCGESCVFIPCISSVLGCSCKNKVCYRN Globa_E 254 GSAFGCGETCVKGKCNTPGCVCSWPVCKKN Globa_C 255 APCGESCVFIPCISAVLGCSCKSKVCYRN Glopa_D 256 GVPCGESCVWVPCTVTALMGCSCVREVCRKD Glopa_E 257 GIPCAESCVWIPCTVTKMLGCSCKDKVCYN Glopa_A 258 GGSIPCIETCVWTGCFLVPGCSCKSDKKCYLN Glopa_B 259 GGSVPCIETCVWTGCFLVPGCSCKSDKKCYLN Glopa_C 260 GDIPLCGETCFEGGNCRIPGCTCVWPFCSKN Co36 261 GLPTCGETCFGGTCNTPGCTCDPFPVCTHD cycloviolacin_T1 262 GIPVCGETCVGGTCNTPGCSCSWPVCTRN cycloviolacin_T2 263 GLPICGETCVGGTCNTPGCSCSWPVCTRN psyle_A 264 GIACGESCVFLGCFIPGCSCKSKVCYFN psyle_B 265 GIPCGETCVAFGCWIPGCSCKDKLCYYD psyle_C 266 KLCGETCFKFKCYTPGCSCSYFPCK psyle_D 267 GIPCGESCVFIPCTVTALLGCSCQNKVCYRD psyle_E 268 GVIPCGESCVFIPCISSVLGCSCKNKVCYRD psyle_F 269 GVIPCGESCVFIPCITAAVGCSCKNKVCYRD vaby_A 270 GLPVCGETCAGGTCNTPGCSCSWPICTRN vaby_B 271 GLPVCGETCAGGTCNTPGCSCTWPICTRN vaby_C 272 GLPVCGETCAGGRCNTPGCSCSWPVCTRN vaby_D 273 GLPVCGETCFGGTCNTPGCTCDPWPVCTRN vaby_E 274 GLPVCGETCFGGTCNTPGCSCDPWPVCTRN Oak6_cyclotide_2 275 GLPICGETCFGGTCNTPGCICDPWPVCTRD Oak7_cyclotide 276 GSHCGETCFFFGCYKPGCSCDELRQCYKN Oak8_cyclotide 277 GVPCGESCVFIPCLTAVVGCSCSNKVCYLN Oak6_cyclotide_1 278 GLPVCGETCFGGTCNTPGCACDPWPVCTRN Cter_C 279 GVPCAESCVWIPCTVTALLGCSCKDKVCYLD Cter_D 280 GIPCAESCVWIPCTVTALLGCSCKDKVCYLN Cter_E 281 GIPCAESCVWIPCTVTALLGCSCKDKVCYLD Cter_F 282 GIPCGESCVFIPCISSVVGCSCKSKVCYLD Cter_G 283 GLPCGESCVFIPCITTVVGCSCKNKVCYNN Cter_H 284 GLPCGESCVFIPCITTVVGCSCKNKVCYND Cter_I 285 GTVPCGESCVFIPCITGIAGCSCKNKVCYIN Cter_J 286 GTVPCGESCVFIPCITGIAGCSCKNKVCYID Cter_K 287 HEPCGESCVFIPCITTVVGCSCKNKVCYN Cter_L 288 HEPCGESCVFIPCITTVVGCSCKNKVCYD Cter_M 289 GLPTCGETCTLGTCYVPDCSCSWPICMKN Cter_N 290 GSAFCGETCVLGTCYTPDCSCTALVCLKN Cter_O 291 GIPCGESCVFIPCITGIAGCSCKSKVCYRN Cter_P 292 GIPCGESCVFIPCITAAIGCSCKSKVCYRN Cter_Q 293 GIPCGESCVFIPCISTVIGCSCKNKVCYRN Cter_R 294 GIPCGESCVFIPCTVTALLGCSCKDKVCYKN vitri_B 295 GVPICGESCVGGTCNTPGCSCSWPVCTTN vitri_C 296 GLPICGETCVGGTCNTPGCFCTWPVCTRN vitri_D 297 GLPVCGETCFTGSCYTPGCSCNWPVCNRN vitri_E 298 GLPVCGETCVGGTCNTPGCSCSWPVCFRN vitri_F 299 GLTPCGESCVWIPCISSVVGCACKSKVCYKD hedyotide_B1 300 GTRCGETCFVLPCWSAKFGCYCQKGFCYRN

In some embodiments, a peptide derived from, comprising, alternatively consisting essentially of, or yet further consisting of angiotensinogen (SEQ ID NO: 6; GenBank accession numbers: BC011519 (mRNA), AAH11519.1 (protein)) is grafted between the termini of the cyclotide backbone. In one embodiment, the peptide comprises or consists essentially of angiotensin I: DRVYIHPFHL (SEQ ID NO: 7) or an equivalent thereof. In another embodiment, the peptide comprises or consists essentially of angiotensin II: DRVYIHPF (SEQ ID NO: 8) or an equivalent thereof. In another embodiment, the peptide comprises or consists essentially of angiotensin III: RVYIHPF (SEQ ID NO: 9) or an equivalent thereof. In another embodiment, the peptide comprises or consists essentially of angiotensin IV: VYIHPF (SEQ ID NO: 10) or an equivalent thereof. In another embodiment, the peptide comprises or consists essentially of angiotensin (1-7) (AT1-7): DRVYIHP (SEQ ID NO: 11) or an equivalent thereof. Nonlimiting examples of equivalents of AT1-7 include DRVYIP (SEQ ID NO: 12), XRVYIE (SEQ ID NO: 13), and XRVYIHE (SEQ ID NO: 14). In another embodiment, the peptide comprises or consists essentially of angiotensin-(1-5) (AT1-5): DRVYI (SEQ ID NO: 15) or an equivalent thereof. In another embodiment, the peptide comprises or consists essentially of angiotensin (1-4) (AT1-5): DRVY (SEQ ID NO: 16) or an equivalent thereof. As used herein, the phrase “grafted between” in this context means that the AT1-7 peptide is covalently attached to the carboxy and amino termini of the cyclotide backbone. In one aspect, residue X in the above sequences represents L-2,3-diaminopropionic acid (L-Dap). In other aspects, residue X represents one or more unnatural amino acids.

In one embodiment, the cyclotide incorporates one or more unnatural amino acids. “Unnatural amino acids” are non-proteinogenic amino acids that either occur naturally or are chemically synthesized. While unnatural amino acids are not on the standard 20-amino acid list, they can be incorporated into a protein sequence. Non-limiting examples of unnatural amino acids include L-2,3-diaminopropionic acid, DL-2,3-diaminopropionic acid, 2,4-diaminobutyric acid, p-methyxyphenylalanine, p-azidophenylalanine, L-(7-hydroxycoumarin-4-yl)ethylglycine, acetyl-2-naphthyl alanine, 2-naphthyl alanine, 3-pyridyl alanine, 4-chloro phenyl alanine, alloisoleucine, Z-alloisoleucine dcha salt, allothreonine, 4-Iodo-phenylalanine, L-benzothienyl-D-alanine OH.

In some aspects, the cyclotide comprises at least an unnatural amino acid residue but retains six cysteine residues that form three disulfide bonds in a cyclized cyclotide. In one aspect, the unnatural amino acid comprises one or more selected from L-2,3-diaminopropionic acid (L-Dap), p-methyxyphenylalanine, p-azidophenylalanine or L-(7-hydroxycoumarin-4-yl)ethylglycine.

In one aspect, the amino acid sequence of the cyclotide with the grafted AT1-7 peptide comprises or consists essentially of GGVCPKILQRCRRDSDCPGACICRGNGYCGSGDRVYIHP (MCo-AT1-7, SEQ ID NO: 17) or an equivalent thereof. In another aspect, the amino acid sequence of the cyclotide with the grafted AT1-7 peptide comprises or consists essentially of GGVCPKILQRCRRDSDCPGACICRGNGYCGSGDRVYIP (MCo-AT1-7, SEQ ID NO: 18) or an equivalent thereof. In one aspect the amino acid sequence of the cyclotide with the grafted AT1-7 peptide comprises or consists essentially of GGVCPKILQRCRRDSDCPGACICRGNGYCGSGXRVYIE (MCo-AT1-7, SEQ ID NO: 19) or an equivalent thereof. In one aspect, the amino acid sequence of the cyclotide with the grafted AT1-7 peptide comprises or consists essentially of GGVCPKILQRCRRDSDCPGACICRGNGYCGSGXRVYIHE (MCo-AT1-7, SEQ ID NO: 20) or an equivalent thereof. In one aspect, residue X in the above sequences represents L-2,3-diaminopropionic acid (L-Dap). In other aspects, residue X represents one or more unnatural amino acids. In further aspect, residue B in the above sequences represents asparagine or aspartic acid.

In a further aspect, the cyclotides, peptides, and polypeptides of the present disclosure further comprise, or alternatively consist essentially of, or yet further consist of one or more labels or purification markers. In some aspects, the label is detectable and comprises one or more of a fluorescent label, a phosphorescent label, an enzymatic label, a chemical label, or a radiolabel. In some aspects, the label comprises a radiolabel such as ³²P, ³⁵P, ³⁵S, or ¹²⁵I. In other aspects, the label comprises a biotin label, a digoxigenin label, an alkaline phosphatase label, a horse radish peroxidase label, or a fluorescent moiety. In other aspects, the cyclotides, peptides and polypeptides of the present disclosure further comprise, or alternatively consist essentially of, or yet further consist of a purification marker such as an epitope tag (e.g. a histidine tag, FLAG tag, Myc tag, or V5 tag). In a yet further aspect the cyclotides, peptides, and polypeptides of the present disclosure further comprise, or alternatively consist essentially of, or yet further consist of, a carrier, such as a pharmaceutically acceptable carrier.

Further provided is a plurality of cyclotides as described herein, wherein the amino acid sequences of the plurality are the same or different from each other, that are optionally labeled or comprise a purification marker. In a further aspect, the cyclotide or plurality of cyclotides further comprises a pharmaceutically acceptable carrier. The cyclotide or plurality thereof can be combined with a therapeutic agent.

In another aspect, this disclosure provides an isolated polynucleotide encoding one or more of the isolated peptides and/or cyclotides described above, alone or in a replication or expression vector, e.g., a viral vector or a plasmid. The polynucleotide units further contain the necessary regulatory element operatively linked to the coding sequences for expression of the polynucleotide in a host cell. Thus, this disclosure also provides an isolated host cell comprising the recombinant peptide as described above or the recombinant polynucleotide, or vector containing same, also as described above. The isolated host cell is a prokaryotic or a eukaryotic cell. In one particular aspect, the host cell is an E. coli cell. The polynucleotides or peptide can also be chemically synthesized using methods known in the art and described herein.

Further provided is a method for recombinantly producing the peptides of this disclosure by growing an isolated host cell as described above under conditions that favor the expression of the polynucleotide. In one aspect, the peptides are isolated from the host cells. The peptides and polynucleotide can also be chemically synthesized.

“Host cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Examples of such include, eukaryotic cells and prokaryotic cells such as E. coli cells. Examples of eukaryotic cells are provided herein and include, but are not limited to cells from plants, fungi, and animals, e.g., murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. In some aspects, the host cell is a yeast cell such as saccharomyces cerevisiae. The cells can be cultured cells or they can be primary cells. Cultured cell lines can be purchased from vendors such as the American Type Culture Collection (ATCC), U.S.A.

In a further aspect, the polynucleotides, vectors and/or host cells further comprise a label. In a yet further aspect the polynucleotides, vectors and/or host cells further comprise, or alternatively consist essentially of, or yet further consists of, a carrier, such as a pharmaceutically acceptable carrier.

Compositions

Compositions are further provided. The compositions comprise a carrier and one or more of an isolated polynucleotide of the disclosure, an isolated polypeptide of the disclosure, an antibody, a gene delivery vehicle of the disclosure or an isolated host cell of the disclosure. The carriers can be one or more of a solid support or a pharmaceutically acceptable carrier. The compositions can further comprise an adjuvant or other components suitable for administrations as vaccines. In one aspect, the compositions are formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants. In addition, embodiments of the compositions of the present disclosure include one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure, formulated with one or more pharmaceutically acceptable auxiliary substances.

Pharmaceutical formulations and unit dose forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infections, and therapies in which the patient self-administers the drug. In one aspect, the formulation is specific for pediatric administration.

The disclosure provides pharmaceutical formulations in which the one or more of an isolated peptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, can be formulated into preparations for administration in accordance with the disclosure by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives or other anticancer agents. For intravenous administration, suitable carriers include physiological saline, or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringeability exists.

Aerosol formulations provided by the disclosure can be administered via inhalation and can be propellant or non-propellant based. For example, embodiments of the pharmaceutical formulations of the disclosure comprise a peptide of the disclosure formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. A non-limiting example of a non-propellant is a pump spray that is ejected from a closed container by means of mechanical force (i.e., pushing down a piston with one's finger or by compression of the container, such as by a compressive force applied to the container wall or an elastic force exerted by the wall itself (e.g. by an elastic bladder)).

Suppositories of the disclosure can be prepared by mixing a compound of the disclosure with any of a variety of bases such as emulsifying bases or water-soluble bases. Embodiments of this pharmaceutical formulation of a compound of the disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the disclosure. Similarly, unit dosage forms for injection or intravenous administration may comprise a compound of the disclosure in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

Embodiments of the pharmaceutical formulations of the disclosure include those in which one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure is formulated in an injectable composition. Injectable pharmaceutical formulations of the disclosure are prepared as liquid solutions or suspensions; or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles in accordance with other embodiments of the pharmaceutical formulations of the disclosure.

In an embodiment, one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a gene delivery vehicle or vector of the disclosure, or an isolated host cell of the disclosure is formulated for delivery by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery of a compound of the disclosure can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. In some embodiments, a compound of the disclosure is in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.

Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.

Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT Publication No. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).

In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted herein, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.

Suitable excipient vehicles for a peptide of the disclosure are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the compound adequate to achieve the desired state in the subject being treated.

Compositions of the present disclosure include those that comprise a sustained-release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. After administration, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.

In another embodiment, the peptide (as well as combination compositions) is delivered in a controlled release system. For example, a peptide of the disclosure may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, polymeric materials are used. In yet another embodiment a controlled release system is placed in proximity of the therapeutic target, i.e., the liver, thus requiring only a fraction of the systemic dose.

In another embodiment, the compositions of the present disclosure (as well as combination compositions separately or together) include those formed by impregnation of a peptide described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the instant disclosure.

The cyclotides of this disclosure are useful in a variety of in vitro and in vivo methods. In one aspect, the cyclotide is administered to a subject in need thereof for the treatment of lung cancer, breast cancer, and other cancers or tumors that express MAS1. The cyclotides also can be used for vasodilation, anti-proliferative properties and anti-angiogenic properties. The cyclotides also are able to reduce serum-stimulated growth of human lung cancer cells both in vitro and in vivo through activation of the unique AT1-7 receptor, MAS1. Thus, another aspect, the cyclotides are useful to activate MAS1 in vitro and in vivo.

The disclosed above methods comprising contacting the cell or tissue with an effective amount of one or more of: the cyclotide as described herein, the isolated polynucleotide described herein or the host cell described herein. The activation of the MAS1, vasodilation, anti-proliferation of cells, and anti-angiogenic properties, and reduction of the growth of lung cancer cells or tumors can be detected by methods known in the art and described herein. The activation of downstream targets of AT1-7 receptor may also be tested to determine the activation of MAS1. The contacting of the cell or tissue may be in vitro in tissue culture or in vivo in a subject.

A further method aspect relates to a method for reducing or inhibiting metastasis, angiogenesis, treating myocardial infarction and/or inhibiting tumor growth in a subject in need thereof comprising administering an effective amount of one or more of: the polypeptide as described herein, the isolated cyclotide, the polynucleotide described herein or the host cell as described herein to the subject. Inhibition or metastasis, angiogenesis, and tumor growth may be demonstrated by assays known in the art. For example, the inhibition may be demonstrated by the reduction of pro-angiogenic or pro-metastatic markers, the increase in anti-angiogenic or anti-metastatic factors, the reduction in tumor size, or the lack of new tumor growth.

Also provided is a method for promoting tumor cell death, e.g., lung or breast tumor death, in an animal, e.g., mammal in need thereof comprising administering an effective amount of one or more of: the polypeptide as described herein, the isolated polynucleotide described herein or the host cell described herein to the subject. Cell death of a tumor can be measured by a reduction in tumor growth or an increase in markers for cell death, necrosis, or apoptosis.

The compositions can be administered to an animal or mammal by a treating veterinarian or to a human patient by a treating physician.

Having described the general concepts of this invention, the following illustrative examples are provided.

Experimental Examples

In order to produce a novel cyclotide with MAS1 agonistic activity, the cyclotide MCoTI-I was used as molecular cyclotide backbone or scaffold (FIG. 1). MCoTI-cyclotides are found in the dormant seeds of the plant Momordica cochinchinensis, and are potent trypsin inhibitors (Ki about 25 pM). Natively folded MCoTI-cyclotides can be easily produced by standard recombinant methods as well as by chemical synthesis, and can also be easily engineered to incorporate novel biological functions. In addition, MCoTI-cyclotides show very little toxicity to human cells (IC50>100 μM) and therefore represent a desirable molecular scaffold for engineering new compounds with unique biological properties.

To engineer the cyclotide MCoTI-I to have AT1-7 activity, a derivative of the peptide AT1-7 peptide was grafted onto the cyclotide scaffold using loop 6 (FIG. 1). This loop has been shown previously to be more disordered in solution [9] and amenable to sequence variation. The peptide was grafted using the side-chains of residues 1 and 7. For this purpose the original residues at Asp1 and Pro7 in the AT1-7 peptide were replaced by diaminopropionic acid and glutamic acid, respectively. These positions have been shown to tolerate mutations in angiotensin-peptides without affecting their biological activity. For example Asp1 and Pro7 have been replaced by glutamine and cysteine without negatively affecting the biological activity of the corresponding angiotensin-derived peptides. The AT1-7 derived peptide was grafted into the cyclotide backbone between residues Gly1 and Ser32 using the β-amino and γ-carboxylic groups through the creation of two isopeptide bonds (FIG. 1). This allowed keeping the native positive and negative charged groups at the N- and C-terminal of the grafted AT1-7 derivative. The resulting grafted cyclotide was called MCo-AT1-7 (FIG. 1).

Cyclotide MCo-AT1-7 was chemically synthesized using Fmoc-based solid-phase peptide synthesis on a sulfonamide resin. Activation of the sulfonamide linker with iodoacetonitrile, followed by cleavage with ethyl mercaptoacetate and acidolytic deprotection, provided the fully deprotected linear peptide α-thioester (FIG. 2A). The corresponding peptide thioester precursor was efficiently cyclized and folded in a one-pot reaction using sodium phosphate buffer at pH 7.2 in the presence of 1 mM GSH. The cyclization/folding reaction was complete in 24 h (FIG. 2A). The cyclization/folding yields was estimated by HPLC to be about 35% (FIG. 2A). Folded MCo-AT1-7 was purified by reverse-phase HPLC and characterized by ES-MS confirming ≧95% purity (FIG. 2B). Cyclotide MCo-AT1-7 was also characterized by 1H-NMR indicating that adopts a native cyclotide fold (FIG. 3, and FIGS. 8A-8B, and Table 2).

TABLE 2 Tabulation of chemical shifts of backbone amide protons (δ¹H-Nα and δ¹Hα) protons of MCo-AT1-7 and their respective differences from cyclotide MCoTI-I. δ¹H-Nα δ¹Hα Δδ¹H-Nα Δδ¹Hα Residue* (ppm) (ppm) (ppm) (ppm) G1 8.112 3.988 0.001 0.021 G2 8.008 4.002 −0.034 0.169 V3 8.73 4.013 0.346 0.138 C4 8.665 5.13 0.083 0.064 P5 N/A — N/A — K6 8.138 4.177 −0.004 0.046 I7 7.541 4.177 −0.085 −0.081 L8 8.412 4.411 −0.177 0 Q9 8.602 4.267 −0.187 −0.195 R10 8.601 4.689 0.075 0.491** C11 8.185 4.71 −0.12 −0.043 R12 9.289 4.303 0.018 −0.019 R13 7.987 4.645 0.028 0 D14 9.065 3.956 −0.071 −0.029 S15 8.088 4.15 0.03 −0.021 D16 7.642 4.436 0.006 −0.07 C17 7.815 4.807 −0.194 −0.116 P18 N/A — N/A — G19 8.414 3.685 0.022 0.063 A20 8.412 4.314 0.089 −0.071 C21 7.994 4.478 −0.184 0.036 I22 8.899 4.289 −0.042 −0.021 C23 9.275 4.828 0.237 0.005 R24 8.013 4.171 0.064 −0.018 G25 8.975 3.813 0.165 0.011 N26 7.685 4.693 −0.014 0.113 G27 8.261 3.869 −0.061 0.01 Y28 7.202 5.127 0.028 −0.017 C29 8.668 5.29 −0.033 0.015 S30 9.685 3.842 −0.043 0 S31 8.672 4.468 −0.02 0.095 G32 8.9 4.008 −0.141 −0.302 *Sequence numbers are based on McoTI-I. **A rather large chemical shift difference (~0.5 ppm) of R10 alpha proton between MCo-AT1-7 and MCoTI-I is possibly induced by the concomitant changes in C11-C23 disulfide bridge related to grafting.

The biological stability of cyclotide MCo-AT1-7 was compared to that of the empty scaffold (MCoTI-I) and the peptide AT1-7 (FIG. 4). This was accomplished by incubating the corresponding peptides in human serum at 37° C. The quantitative analysis of undigested polypeptides was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Naturally occurring MCoTI-cyclotides present a very rigid structure, which makes them extremely stable to proteolytic degradation. Remarkably, cyclotide MCo-AT1-7 was only slightly less stable (τ_(1/2)=39±5 h) than the parent cyclotide MCoTI-I (τ_(v2)=55±7 h) (FIGS. 4 and 7). In contrast, peptide AT1-7 was degraded considerably faster under the same conditions (τ_(1/2)=44±3 min) (FIGS. 4 and 7). A linearized, reduced and alkylated version of MCo-AT1-7 (FIG. 6) was also rapidly degraded (τ_(1/2)=57±5 min) (FIGS. 4 and 7) indicating the importance of the circular Cys-knot topology for proteolytic stability.

Next, the ability of cyclotide MCo-AT1-7 to activate the MAS1 receptor was tested using CHO cells stably transfected with pTEJ-8 vector expressing recombinant human MAS1 in a cell-based fluorescence assay to detect the amount of NO release (FIG. 5). Cyclotide MCo-AT1-7 and peptide AT1-7 (used as positive control) were able to increase the intracellular concentration of NO in a dose dependent manner as measured by the level of fluorescence. The naturally-occurring cyclotide MCoTI-I did not show any increase in intracellular NO levels (FIG. 5). When the MAS1 activation biological assay was performed in the presence of the MAS1 peptide antagonist A779, no increase in the intracellular concentration of NO was detected therefore confirming the agonistic activity of the cyclotide MCo-AT1-7. A similar profile was obtained with the peptide AT1-7 (FIG. 5). The activity of the cyclotide MCo-AT1-7 was estimated to be about 90% of that of peptide AT1-7 at 10 nM in this assay.

In summary report here for the first time the design and synthesis of a novel cyclotide able to efficiently activate the MAS1 receptor. This was successfully accomplished by grafting an AT1-7-derived peptide onto loop 6 of the cyclotide MCoTI-I using the side-chains of the first and last residues of the grafted peptide through the formation of isopeptide bonds (FIG. 1). 1H-NMR studies also revealed that the grafting of the AT1-7-derived peptide using isopeptide bonds onto this loop did not affect the native cyclotide scaffold, indicating the tolerance of this loop for the grafting of peptide sequences using non-native peptide bonds. Cyclotide MCo-AT1-7 showed itself to be a potent MAS1 agonist, with similar activity to that of the peptide AT1-7. In addition, cyclotide MCo-AT1-7 showed a remarkable resistance to biological degradation in human serum, with a τ½ value of about 39 h. This value is similar to that of the cyclotide MCoTI-I and significantly higher that the half-life of the AT1-7 peptide (τ_(1/2) about 1 h).

These results show that engineered cyclotides hold great promise for the development of a novel type of peptide-based therapeutic able to efficiently target extracellular protein/protein interactions. The results demonstrate for the first time the design of an engineered cyclotide using isopeptide peptide bonds able to activate the MAS1 receptor with low nanomolar activity and very high serum stability, thereby providing a promising lead compound for the design of novel therapeutics for the treatment of cancer and myocardial infarction.

Materials and Methods

All chemicals involved in synthesis or analysis were obtained from Aldrich (Milwaukee, Wis.) or Novabiochem (San Diego, Calif.) unless otherwise indicated. Analytical HPLC was performed on a HP1100 series instrument with 220 and 280 nm detection using a Vydac C18 column (5 micron, 4.6×150 mm) at a flow rate of 1 mL/min. Preparative and semipreparative HPLC were performed on a Waters Delta Prep system fitted with a Waters 2487 UV-visible detector using either a Vydac C18 column (15-20 μm, 50×250 mm) or a Vydac C18 (15-20 μm, 10×250 mm) at a flow rate of 50 or 5 mL/min, respectively. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA) (solvent A) vs. 0.1% TFA, 90% acetonitrile in H2O (solvent B). Ultraviolet-visible (UV-vis) spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer. Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all compounds and components of reaction mixtures. ES-MS was performed on an Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer using Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2.

Preparation of Fmoc-Tyr(tBu)-F

For all peptides synthesis, Fmoc-Tyr(tBu)-F was prepared using dimethylaminosulfur trifluoride (DAST) and immediately loaded to resin as described in Kluskens et al. J Pharmacolol Exp Ther 2009, 328, 849-854, incorporated herein by reference. Briefly, DAST (160 μL, 1.2 mmol) was added drop wise at 25° C. under nitrogen current to a stirred solution of Fmoc-Tyr(tBu)-OH (459.6 mg, 1 mmol) in 10 mL of dry dichloromethane (DCM), containing dry pyridine (81 μL, 1 mmol). After 20 minutes, the mixture was washed with ice-cold water (3×20 mL). The organic layer was separated and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to give the corresponding Fmoc-amino acyl fluoride as yellowish oil that was immediately used.

Loading of 4-Sulfamylbutyryl AM Resin with Fmoc-Tyr(tBu)-F

Loading of the first residue for all peptides was accomplished using Fmoc-Tyr(tBu)-F according to standard protocols (e.g. Contreras et al. J. Control. Release 2011, 155, 134-143). Briefly, 4-Sulfamylbutyryl AM resin (420 mg, 0.33 mmol) (Novabiochem) was swollen for 30 minutes with dry DCM and then drained. A solution of Fmoc-Tyr(tBu)-F (about 461 mg, 1 mmol) in dry DCM (2 mL) and di-isopropylethylamine (DIEA) (180 μL, 1 mmol) was added to the drained resin and reacted at 25° C. for 1 h. The resin was washed with dry DCM (5×5 mL), dried and kept at −20° C. until use.

Chemical Synthesis of Cyclotide MCo-AT1-7

Solid-phase synthesis of cyclotide MCo-AT1-7 was carried out on an automatic peptide synthesizer ABI433A (Applied Biosystems) using the Fast-Fmoc chemistry with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/diisopropylethylamine (DIEA) activation protocol at 0.1 mmole scale on a Fmoc-Tyr(tBu)-sulfamylbutyryl AM resin. Side-chain protection compatible with Fmoc-chemistry was employed as previously described for the synthesis of peptide a-thiesters by the Fmoc-protocol (see, e.g. Cavanagh et al. J. Magn. Res. 1992, 96, 670-678), except for the N-terminal Cys residue, which was introduced as Boc-Cys(Trt)-OH. Following chain assembly, the alkylation, thiolytic cleavage and side chain deprotection were performed as described in Contreras et al. J. Control. Release 2011, 155, 134-143. Briefly, about 100 mg of protected peptide resin were first alkylated two times with ICH2CN (174 μL, 2.4 mmol; previously filtered through basic alumina) and DIEA (82 μL, 0.46 mmol) in N-methylpyrrolidone (NMP) (2.2 mL) for 24 hours. The resin was then washed with NMP (3×5 mL) and DCM (3×5 mL). The alkylated peptide resin was cleaved from the resin with HSCH2CO2Et (200 μL, 1.8 mmol) in the presence of a catalytic amount of sodium thiophenolate (NaSPh, 3 mg, 22 μmol) in dimethylformamide (DMF):DCM (1:2 v/v, 1.2 mL) for 24 hours. The resin was then dried at reduced pressure. The sidechain protecting groups were removed by treating the dried resin with trifluoroacetic acid (TFA):H2O:triisopropylsilane (TIS) (95:3:2 v/v, 10 mL) for 3-4 h at room temperature. The resin was filtered and the linear peptide thioester was precipitated in cold Et2O. The crude material was dissolved in the minimal amount of H2O:MeCN (4:1) containing 0.1% TFA and characterized by HPLC and ES-MS [Expected mass: 4361.1 Da. Observed mass: 4360.0±0.7 Da] as the desired grafted MCoTI-I linear precursor α-thioester (FIG. 2). Cyclization and folding was accomplished by flash dilution of the linear α-thioester TFA crude to a final concentration of about 50 μM into freshly degassed 1 mM reduced glutathione (GSH), 0.1 M sodium phosphate buffer at pH 7.2 for 24 h (FIG. 2). The folded cyclotide was purified by semi-preparative HPLC using a linear gradient of 17-28% solvent B over 30 min. The purified cyclotide was characterized by HPLC and ES-MS confirming ≧95% purity (FIG. 2).

Chemical Synthesis of Linearized and S-Alkylated Cyclotide MCo-AT1-7

For the preparation of linearized and S-alkylated MCo-AT1-7, 20 mg of sulfonamide peptideresin was activated as described above and cleaved from the resin with propylamine in DMF (1:1, 0.8 mL) for 4 h. The resulting peptide was side deprotected and peptide collected by precipitation with diethyl ether as described above. The linear peptide was fully reduced with dithiothretol (40 mM, 300 μL) in freshly degassed 0.1 M Na₂HPO₄ buffer at pH 7.5 at 37° C. for 1 h followed by alkylation of all free cysteines with iodoacetamide (0.5 M, 200 μL) for 10 min at room temperature.

The linear and fully reduced cyclotide precursor was purified by semi-preparative HPLC using a linear gradient of 15-35% solvent B over 30 min. The purified peptide was characterized by HPLC and ES-MS [Expected mass: 4641.2 Da. Observed mass: 4641.1±0.6 Da] (FIG. 6).

NMR Spectroscopy

NMR samples were prepared by dissolving cyclotides into 80 mM potassium phosphate pH 6.0 in 90% H₂O/10% ²H₂O (v/v) to a concentration of approximately 0.5 mM. All ¹H NMR data were recorded on Bruker Avance II 700 MHz spectrometer equipped with the TXI cryoprobe. Data were acquired at 298 K, and 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, was used as an internal reference. The carrier frequency was centered on the water signal, and the solvent was suppressed by using WATERGATE pulse sequence. ¹H, ¹HTOCSY (spin lock time 80 ms) and ¹H, ¹H-NOESY (mixing time 150 ms) spectra were collected using 4096 t₂ points and 256 t₁ of 64 transients. Spectra were processed using Topspin 2.1 (Bruker). Each 2D-data set was apodized by 90°-shifted sinebell-squared in all dimensions, and zero filled to 4096×512 points prior to Fourier transformation. Assignments for H^(a) (H-C^(a)) and H′ (H-N^(a)) protons of folded MCo-AT1-7 (Table 2) were obtained using standard procedures.

Biological Activity of Cyclotide MCo-AT1-7

CHO cells stably transfected with pTEJ-8 vector expressing recombinant human MAS1 clone are grown to confluency. After washing, cells are incubated for a short time in 700 μL of supplemented Tyrode's salts containing 10 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), 100 μM 2,3-diaminonaftalene (DAN), and 1 mM L-arginine. When using antagonist for competition assays, cells are exposed to MAS1 antagonist A779 (D-Ala⁷-AT 1-7) at 0.1 μM for 15 min before addition of the cyclotides to be tested. Different concentrations of the cyclotides to be analyzed are added to the cell medium and the plates are agitated for 1 min before being placed into the incubator for 2 hours. After 2 hours, the cellular supernatants are transferred to opaque 96 well plates, and the amount of released NO is measured by fluorescence (λex=380 nm, λem=425 nm).

Serum Stability

Human serum was spun down at 15,000 rpm for 10 min to separate the lipid components. Peptides (about 15 μg dissolved in 15 μl PBS) were mixed with 150 μl human serum and incubated in a 37° C. water bath. Aliquot samples (15 μL) were taken at different time points and serum protein were precipitated with about 70% MeCN at 4° C. for 10 min. The supernatant was separated, lyophilized, dissolved in 5% MeCN in water containing 0.1% formic acid, and analyzed by HPLC-MS/MS.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Several aspects of the invention are listed below.

Cyclotide Treatment of Lung Cancer In Vitro

To test the effectiveness of the cyclotides and peptides disclosed herein in the treatment of cancer, human adenocarcinoma cell lines such as SK-LU-1 and A549, as well as non-small lung cancer cell lines such as SK-MES-1 will treated be treated with serum containing MCo-AT1-7 or a control MCo-TI-I cyclotide. Serum-stimulated growth of the cell lines treated with cyclotides will be compared. A MCo-AT1-7 dose and/or time specific reduction in growth or increase in cell death of one or more cell lines will indicate that the cyclotide has a significant inhibitory effect. Cells treated with an effective amount of MCo-AT1-7 may exhibit reduced DNA synthesis, reduced cell number, increased apoptosis, reduced metabolic activity, and/or a reduction in proliferation (as measured by cell cycle analysis) relative to cells treated with the control cyclotide.

For example, to measure the effect of MCo-AT1-7 on lung cancer cell proliferation, a growth assay will be performed. Cyclotides with AT 1-7 grafts will be prepared according to methods disclosed herein. SK-LU-1, A549, and/or SK-MES-1 will be seeded into tissue culture plates in the presence of about 1% FBS. Every 12 or every 24 hours following initial plating, cells will be treated with about 100 pM, about 500 pM, about 1 nM, about 10 nM, about 20 nM, about 50 nM, about 100 nM, about 200 nM, about 500 nM, about 800 nM, about 1 μM, about 2 μM, about 10 μM MCo-AT1-7 or control cyclotide in phosphate-buffered saline or PBS alone. Cells will be removed and counted on days 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, and 18 using trypsin with EDTA and a hemocytometer. Additionally, cells may be pre-treated with cyclotides in the growth medium for about 30 minutes, about an hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours prior to the addition of serum in order to test chemopreventative effects of MCo-AT1-7. To measure cell death, dye uptake and/or Annexin V assays will be performed on harvested cells. Additionally, BrdU incorporation assays coupled with flow cytometry and/or immunofluorescence will used to measure cell proliferation in cells treated with cyclotides as described above. To measure the levels of DNA synthesis, a thymidine incorporation assay will be used on quiescent cells. Tritiated thymidine will be added to the growth medium of semi-confluent cells treated with serum and cyclotides as above. After about 4 hours of thymidine exposure, the level of incorporated thymidine will be determined by liquid scintillation spectrometry. The results of these in vitro studies will indicate that MCo-AT1-7 inhibits lung cancer cell growth and validate MCo-AT1-7 as a novel chemopreventive and/or chemotherapeutic treatment for lung cancer.

Cyclotide Treatment of Lung Cancer In Vivo

A human lung tumor xenograft model will be used to test the inhibitory effects of the MCo-AT1-7 on lung cancer in vivo. Actively proliferating human lung cancer cells (e.g. SK-LU-1, A549, and/or SK-MES-1) will be inoculated into athymic mice. The dose of cancer cells may vary by cell line but will be about 1×10² cells, about 1×10³ cells, about 1×10⁴ cells, about 1×10⁵ cells, about 1×10⁶ cells, about 1×10⁷ cells, about 1×10⁸ cells, or about 1×10⁹ cells per mouse. After about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, and/or about 48 days, mice will be treated with MCo-AT1-7 or a control MCo-TI-I cyclotide. The number of days prior to initiating cyclotide treatment may be determined based on a threshold minimum tumor size such (e.g. about 10 mm³, 100 mm³, or about 1000 mm³). The cyclotides will be prepared as disclosed herein. Cyclotides may be administered to the mice by any method in the art including, but not limited to, osmotic mini-pump (Durect Corp.), intravenous injection, intraarterial injection, intraperitoneal injection, or inhalation. The dose may vary by method of administration. Non limiting examples of an effective dose for delivery via an osmotic pump are about 100 ng/kg/hr, about 500 ng/kg/hr, about 1 μg/kg/hr, about 10 μg/kg/hr, about 20 μg/kg/hr, about 30 μg/kg/hr, about 50 μg/kg/hr, about 50 μg/kg/hr, 100 μg/kg/hr, or about 1 mg/kg/hr. The pump may also contain heparin (about 25 U/mL) or an equivalent anticoagulant. Chemotherapeutic agents such as cisplatin, carboplatin, paclitaxel, gemcitabine, or docetaxel may be co-administered with the cyclotides. The mice will be anesthetized by inhalation with isoflurane. Administration may occur over a period of about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, or about 24 hours and may be repeated daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or every 14 days. Inhibition of tumor growth will be determined by harvesting tumors from MCo-AT1-7 or control treated mice, measuring tumor size, and performing immunohistochemistry to assay for cancer biomarkers, proliferation/cell cycle markers, cell death markers, and/or human cell markers. Prior to sacrificing mice, the mice may be administered BrDU to perform cell cycle assays on the tumor cells. Tumors from mice treated with MCo-AT1-7 may be smaller in size, exhibit reduced proliferation, and/or exhibit increased cell death relative to control-treated mice.

It should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention such as for example, embodiments described in Appendix A attached hereto. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

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1. A cyclotide comprising a cyclotide backbone and a peptide comprising an angiotensin polypeptide or an equivalent thereof.
 2. The cyclotide of claim 1, wherein the angiotensin polypeptide comprises a polypeptide of the group of an angiotensin1-1-7 polypeptide, an angiotensin 1-4 polypeptide, or an angiotensin 1-5 polypeptide, or an equivalent of each thereof.
 3. The cyclotide of claim 1, wherein the angiotensin polypeptide is from the group of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 (XRVYIE), wherein X is the amino acid L-2,3-diaminopropionic acid, SEQ ID NO: 14 (XRVYIHE), wherein X is the amino acid L-2,3-diaminopropionic acid, SEQ ID NO: 15, SEQ ID NO: 16, or an equivalent of each thereof.
 4. A cyclotide of claim 1, wherein the cyclotide is from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or an equivalent of each thereof, wherein the equivalent comprises a polypeptide that maintains a cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.
 5. The cyclotide of claim 1, wherein the cyclotide backbone is a polypeptide from the group of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or an equivalent of each thereof, wherein the equivalent comprises a polypeptide that maintains a cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.
 6. The cyclotide of claim 1, wherein the cyclotide backbone is a polypeptide from the group of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, or an equivalent of each thereof, wherein the equivalent comprises a polypeptide that maintains a cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.
 7. The cyclotide of claim 1, further comprising a label or purification marker.
 8. The cyclotide of claim 1, further comprising a carrier.
 9. A plurality of cyclotides of claim
 1. 10. The plurality of claim 9, wherein the amino acid sequences of the plurality are the same or different from each other.
 11. The cyclotide of claim 8, wherein the carrier is a pharmaceutically acceptable carrier.
 12. The plurality of claim 10, further comprising a carrier.
 13. The plurality of claim 12, wherein the carrier is a pharmaceutically acceptable carrier.
 14. The cyclotide of claim 11, wherein the pharmaceutically acceptable carrier further comprises a therapeutic agent.
 15. The plurality of claim 12, wherein the pharmaceutically acceptable carrier further comprises a therapeutic agent.
 16. An isolated polynucleotide encoding the cyclotide of claim 1, or an equivalent of each thereof.
 17. A complement of the polynucleotide of claim
 16. 18. An isolated polynucleotide of claim 17, further comprising a label or a purification marker.
 19. An isolated polynucleotide of claim 16, and a carrier.
 20. The isolated polynucleotide of claim 19, wherein the carrier is a pharmaceutically acceptable carrier.
 21. A vector comprising the isolated polynucleotide of claim
 16. 22. An isolated host cell comprising the cyclotide of claim
 1. 23. The isolated host cell of claim 22, wherein the cell is a eukaryotic cell or a prokaryotic cell.
 24. An isolated host cell comprising the polynucleotide of claim
 16. 25. The isolated host cell of claim 24, wherein the isolated host cell is a eukaryotic cell or a prokaryotic cell.
 26. The isolated host cell of claim 25, wherein the cell is a prokaryotic cell.
 27. A method for producing a recombinant cyclotide polypeptide, comprising growing the isolated host cell of claim 24, under conditions to express the polynucleotide.
 28. The method of claim 27, further comprising purifying the polypeptide.
 29. A method for any one of: promoting vasodilation in a tissue, inhibiting the proliferation of a cell or tissue, or inhibiting angiogenesis, comprising contacting the cell or tissue with a cyclotide of claim
 1. 30. The method of claim 29, wherein the cell or tissue is a mammalian cell or tissue.
 31. The method of claim 30, wherein the mammalian cell or tissue is a human cell or tissue.
 32. The method of 29, wherein the contacting is in vitro or in vivo.
 33. A method for inhibiting the growth of a tumor cell comprising contacting the tumor cell with a cyclotide of claim
 1. 34. The method of claim 33, wherein the tumor cell expresses MAS1.
 35. The method of claim 33, wherein the tumor cell is a lung cancer tumor cell or a breast cancer tumor cell.
 36. The method of claim 33, wherein the tumor cell is mammalian cell.
 37. The method of claim 36, wherein the mammalian cell is a human cell.
 38. The method of claim 33, wherein the contacting is in vitro or in vivo.
 39. A method for any one of: promoting vasodilation in a tissue, inhibiting the proliferation of a cell or tissue, treating myocardial infarction or inhibiting angiogenesis, in a subject in need thereof, comprising administering to a subject in need thereof, an effective amount of cyclotide of claim
 1. 40. A method for inhibiting the growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the cyclotide of claim
 1. 41. The method of claim 40, wherein the tumor expresses MAS1.
 42. The method of claim 41, wherein the tumor is a lung cancer tumor or breast cancer tumor.
 43. The method of claim 40, wherein the subject is a mammal.
 44. The method of claim 40, wherein the subject is a human. 